Functional capacity of alveolar neutrophils in acute respiratory distress syndrome

Abstract

BackgroundInappropriate accumulation or persistence of neutrophils (polymorphonuclear leucocytes [PMNs]), within the alveolar airspace is considered to be crucial to the pathogenesis of acute respiratory distress syndrome (ARDS). Previous work has established that alveolar PMNs from patients with ARDS have a pro-survival phenotype, and that bronchoalveolar lavage fluid (BALF) in ARDS suppresses constitutive PMN apoptosis in vitro. However, the functional activity of alveolar-sequestered PMNs in ARDS is unknown, partly due to the technical challenges in obtaining and purifying alveolar PMNs. Proinflammatory mediators in the ARDS alveolar milieu conceivably prime or activate key PMN functions in vivo, including the generation of toxic reactive oxygen species (ROS). This research evaluates agonist-stimulated ROS generation in alveolar and blood PMNs from the same patient with ARDS compared with healthy volunteer blood PMNs. MethodsAlveolar and blood PMNs from mechanically ventilated patients who fulfilled the Berlin criteria of ARDS were studied in parallel. Patients underwent fibreoptic bronchoscopy and bronchoalveolar lavage within 36 h of ARDS onset, and the recovered BALF was processed immediately. Alveolar PMNs were isolated to 90% (SE 2·5) purity with a rapid semi-automated negative-selection immunomagnetic technique (Robosep, Stemcell Technologies, Vancouver, Canada). BALF supernatants and serum were stored at −80°C for subsequent cytokine analysis. Patients undergoing bronchoalveolar lavage for other clinical indications (sarcoidosis, tracheomalacia, and haemoptysis) served as controls. Blood PMNs were isolated using dextran sedimentation and plasma Percoll gradients. Neutrophil apoptosis was detected by flow cytometry using annexin V and propidium iodide. Kinetics of the PMN oxidative burst in response to ex-vivo stimulation with N-formyl-methionyl-leucylphenylalanine was determined with real-time horseradish peroxidase–luminol-dependent chemiluminescence detection. FindingsThe BALF from patients with ARDS consisted predominantly of alveolar PMNs (65%, SE 5), whereas alveolar macrophages constituted the major cell type (92%, SE 5) in BALF from control individuals. Nearly 70% of ARDS alveolar PMNs showed hypersegmented nuclear morphology and had a reduced rate of apoptosis after 20 h in culture (22% apoptotic PMNs, SE 10) compared with blood PMNs from control individuals (68% apoptotic PMNs, SE 7·2). ROS production by untreated alveolar neutrophils was 1·5 times higher compared with optimally (tumour necrosis factor α) ex vivo-primed ARDS blood PMNs from the same ARDS patient and control blood PMNs. This finding could reflect the alveolar cytokine milieu since concentrations of the proinflammatory mediators interleukin (IL) 1, IL6, IL8, IL10, IL12, and tumour necrosis factor α were significantly increased in BALF and serum from patients with ARDS compared with controls. InterpretationThese findings indicate that ARDS alveolar PMNs have altered morphology, enhanced survival, and augmented capacity for ROS generation, which might contribute to PMN-dependent tissue injury. FundingNIHR Cambridge Biomedical Research Centre, GlaxoSmithKline.